This invention relates to a method of enhancing the efficacy of recombinant insect viruses, such as baculoviruses, for use as insecticides. This invention relates to recombinant insect viruses and vectors for use therewith in which the expression of a heterologous gene or fragments thereof (preferably encoding an insect controlling substance or modifying substance, such as an insect toxin) is operably linked to an early promoter.
The following abbreviations are used throughout this application:
AcMNPVxe2x80x94Autographa californica nuclear polyhedrosis virus
bpxe2x80x94base pairs
BEVSxe2x80x94baculovirus expression vector system
ECVxe2x80x94extracellular virus
GVxe2x80x94granulosis virus
kDxe2x80x94kilodaltons
NPVxe2x80x94nuclear polyhedrosis virus
occxe2x88x92xe2x80x94occlusion negative virus(es)
occ+xe2x80x94occlusion positive virus(es)
OVxe2x80x94occluded virus
PCRxe2x80x94polymerase chain reaction
pfuxe2x80x94plaque forming unit
p.i.xe2x80x94post-infection
PIBxe2x80x94polyhedron inclusion body (also known as occlusion body)
5xe2x80x2 UTR: The mRNA or gene sequence corresponding to the region extending from the start site of gene transcription to the last base or base pair that precedes the initiation codon for protein synthesis.
3xe2x80x2 UTR: The mRNA or gene sequence corresponding to the region extending from the first base or basepair after the termination codon for protein synthesis to the last gene-encoded base at the 3xe2x80x2 terminus of the mRNA.
(+)strand: Refers to the DNA strand of a gene and its flanking sequences which has the same sense as the RNA that is derived from that gene.
(xe2x88x92)strand: Refers to the DNA strand of a gene and its flanking sequences that is complementary to the (+)strand.
Since the advent of recombinant DNA technology, there has been steady growth in the number of systems available for the regulated expression of cloned genes in prokaryotic and eukaryotic cells. One eukaryotic system that has gained particularly widespread use is the baculovirus expression vector system, or BEVS, developed by Smith and Summers (1). This system utilizes a nuclear polyhedrosis virus isolated from the alfalfa looper, Autographa californica, as a vector for the introduction and high level expression of foreign genes in insect cells.
Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) is the prototype virus for the Family Baculoviridae. These viruses have large, circular, double-stranded DNA genomes (at least 90-230 kilobases (2)). There are two Subfamilies, Nudibaculovirinae, which do not form occlusion bodies, and the Eubaculovirinae, which are characterized by their ability to form occlusion bodies in the nuclei of infected insect cells. The structural properties of the occlusion bodies are used to further classify the members of this Subfamily into two genera: the nuclear polyhedrosis viruses (NPVs) and the granulosis viruses (GVs).
As exemplified by AcMNPV, the occlusion bodies formed by NPVs are 1-3 microns in diameter and typically contain several hundred virions embedded in a para-crystalline matrix. Occlusion bodies are also referred to as either polyhedra (polyhedron is the singular term) or as polyhedron inclusion bodies (PlBs). The major viral-encoded structural protein of the occlusion bodies is polyhedrin, which has a molecular weight of 29 kilodaltons (kD) (1,3). More than a hundred such occlusions can frequently be found in the nucleus of a single infected cell. GVs are distinguished from NPVs by the fact that their occlusions are much smaller and contain only one virion, which is embedded in a matrix of the viral protein granulin. Nevertheless, the fundamental principles of GV replication are similar to those described below for AcMNPV.
Viral occlusion bodies play an essential role in the horizontal (insect to insect) transmission of Eubaculovirinae. When a larva infected with AcMNPV dies, large numbers of occlusion bodies are left in the decomposing tissues. In neutral or acidic conditions (pH less than 10), the protein matrix and outer calyx of the occlusion body protect the embedded virions against chemical degradation in the environment and provide limited protection against UV radiation. However, when the occlusion bodies are ingested by another larva, they dissolve rapidly in the larval midgut, which is strongly alkaline (pH 10.5-12), and the embedded virions are released. These virions then adsorb to and infect various types of midgut cells.
Infected midgut cells synthesize few if any new occlusion bodies. Instead, they produce a second form of the virus, known as extracellular virus (ECV). Whereas the occluded form of the virus is responsible for the horizontal transmission of the virus among larvae, the ECV is used to spread the infection from tissue to tissue internally. This is an essential aspect of normal viral pathogenesis and continues until most tissues of the larva have been infected and lysed. As the virus spreads internally, many of the infected cells, especially hemocytes and fat body cells, produce not only more ECV, but also copious amounts of occluded virus (OV) in the form of occlusion bodies. When the larva dies, the occlusion bodies are deposited in the environment and the cycle begins anew.
Although ECV and OV are genetically identical, they are biochemically distinct. Shortly after the AcMNPV infects a cell, the nucleocapsid structure (which contains the DNA genome) migrates to the nucleus of the cell, where it is uncoated. This sets in motion a regulated cascade of viral gene expression which leads to the onset of viral DNA synthesis (at about 6-12 hours post-infection (p.i.)) and the formation of many new nucleocapsids. ECV production begins at about 10-13 hours p.i. with the budding of the nucleocapsids through the cytoplasmic surface of the cell. During the budding process, the nucleocapsids acquire a lipid membrane, or envelope, which is decorated with a viral glycoprotein known as gp64. This protein is specific to the ECV form of the virus and is required for ECV infectivity. The formation of occlusion bodies begins much later (24-36 hours p.i.) and requires the concerted action of numerous specialized viral gene products, the most prominent of which is polyhedrin.
The polyhedrin gene plays a central role in the BEVS technology. Because large amounts of polyhedrin are required for occlusion body formation, the polyhedrin gene is one of the most actively transcribed genes in the viral genome during the very late phases of virus replication. Smith and Summers (1) show that expression of a heterologous gene can be achieved by substituting the coding region of the polyhedrin gene with the coding region of a heterologous gene of interest. Since polyhedrin is not required for ECV formation, the resulting virus is able to replicate normally in cultured insect cells. However, it is no longer able to produce polyhedrin for occlusion body formation and is therefore occlusion-negative (occxe2x88x92).
The BEVS has been used successfully to express foreign genes isolated from a wide range of prokaryotic and eukaryotic organisms and viruses. Some representative examples include the human xcex1- and xcex2-interferons, the Drosophila Krueppel gene product, E. coli xcex2-galactosidase, various HIV structural proteins, and a Neurospora crassa site-specific DNA binding protein (3). In general, these genes may encode cytosolic proteins, nuclear proteins, mitochondrial proteins, secreted proteins or membrane-bound proteins. In most cases, the proteins are biologically active and undergo appropriate post-translational modification, including proteolytic processing, glycosylation, phosphorylation, myristylation and palmitylation. Hence, this system has proven to be a highly valued tool for both fundamental molecular research and for the production of proteins for commercial purposes. Using BEVS technology, recombinant viruses are produced in cultured insect cells by homologous DNA recombination between AcMNPV DNA and a plasmid-based transfer (or transplacement) vector containing the heterologous gene of interest under the control of the polyhedrin gene promoter. To facilitate homologous DNA recombination the modified polyhedrin gene of the transfer vector is flanked at each end by several kilobases (2-4 kb is typical) of native AcMNPV DNA. Many transfer vectors conforming to this general specification have been described.
In a typical experiment, purified AcMNPV DNA and transfer vector DNA are mixed together and then transfected into Sf9 insect cells. Once the DNA reaches the cell nucleus, it can be acted upon by cellular proteins involved in the transcription, replication, topological management and repair of DNA. Most of the viral DNA is used without modification as a substrate for viral replication; however, a small fraction (typically 0.1-5%) undergoes homologous recombination with the transfer vector prior to the onset of virus replication. The product of this recombination event is a virus in which the wild-type polyhedrin gene has been transplaced by the desired heterologous gene of the transfer vector. These recombinant viruses can be identified visually with low magnification light microscopy as occxe2x88x92 plaques in a standard viral plaque assay.
Modification of the original BEVS technology have been described which allow the construction of recombinant viruses in which the heterologous gene is linked to appropriate regulatory sequences (e.g. promoters and signal peptides, etc.) and inserted into a site which does not disrupt the polyhedrin gene. Such viruses are able to form orally infectious polyhedra, which is generally preferred at present for a commercial insecticide. One drawback of the application of a naturally-occurring insect virus, such as a baculovirus, as a pesticide is the time required for inactivation or death of an insect, particularly when compared with chemical insecticides. Typically, insect viruses such as baculoviruses can take from 4 to 5 days to 2 weeks to kill a susectible insect, during which time the insect continues to feed and damage crops. In order to increase the activity of the insect viruses, heterologous genes producing insect controlling substances, such as toxins, have been introduced into the insect virus to enhance its speed of action on target insects. Prior to the present invention, recombinant insect viruses with enhanced speed of action against target insects contained a heterologous gene under the control of a baculovirus late or very late gene promoter, such as polyhedrin gene promoter or the p10 promoter. Such promoters were selected because they are derived from genes that are abundantly transcribed during the baculovirus life cycle. The invention provided herein further increases the efficacy (speed of action) of the recombinant insect virus upon infection of an insect cell through the use of viral early promoters.
It is an object of the present invention to construct recombinant DNA insect viruses, such as baculoviruses, and vectors for insect viruses useful for expression of a heterologous gene encoding an insect controlling substance or modifying substance, in which the expression of such a gene is operably-controlled by an early promoter. It is also an object of this invention to provide a method of expressing an insect controlling or insect modifying substance in insect cells comprising infecting insect cells with a recombinant insect virus of this invention.
It is also an object of this invention to provide an expression cassette, which comprises a gene sequence comprising the heterologous gene and an early promoter, which is operably linked to said heterologous gene for expression. Once the expression cassette is operably placed in an insect virus, the heterologous gene is expressible from the recombinant insect virus upon infecting an insect cell.